In the early 2000s, researchers first proposed that diffusible small-molecule signals among members of the same micro-community were critical for the regulation of gene responses and plaque maturation. These signals were later determined to correspond to AHLs (1). As a major class of AI signals produced by Proteobacteria, AHLs commonly comprise a homoserine lactone ring carrying an acyl chain of C₄–C₁₈ in length (14). A rather short-chain-length AHL, N-octanoyl-L-homoserine lactone (C₈-HSL), was isolated from a laboratory monospecies sample of the oral bacteria Porphyromonas gingivalis (P. gingivalis). Meanwhile, multi-species oral biofilms formed by Streptococcus oralis, Veillonella parvula, Actinomyces naeslundii, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and P. gingivalis cultures contain small amounts of QS signalling N-3-oxo-octanoyl-homoserine lactone (OC₈-HSL) (15).

Several AHLs have been previously described in bacteria clinically isolated from human oral samples and identified using methods such as high-resolution mass spectrometry (16). Bacterial strains, such as Enterobacter spp., taken from the human tongue surface were found to produce C₈-HSL and N-dodecanoyl-homoserine lactone (C₁₂-HSL) (17, 18). Furthermore, N-hexanoyl-L-homoserine lactone (C₆-HSL), C₈-HSL, N-decanoyl-L-homoserine lactone (C₁₀-HSL), and C₁₂-HSL were identified from dentin caries (19). Additionally, N-butyryl-L-HSL (C₄-HSL), C₆-HSL, C₈-HSL, and N-hexadecanoyl-L-homoserine lactone (C₁₆-HSL) were identified in bacteria isolated from a dental plaque biofilm sample (20).

The OC₈-HSL was mostly found in saliva samples from healthy donors (21). It was also isolated from teeth extracted from healthy individuals, patients with dental cavities, and individuals with gastrointestinal disease (4). N-tetradecanoyl-L-homoserine lactone (C₁₄-HSL) and N-octadecanoyl-L-homoserine lactone (C₁₈-HSL) have been detected in the saliva of patients with dental caries. C₈-HSL and occasionally C₁₄-HSL have been found in patients with periodontal disease (21) (Table 1). Overall, AHLs' presence in both laboratory and clinical samples underscores their significance as an integral role in the communication and coordinated behaviour of bacterial communities in the oral cavity, suggesting their broad involvement in oral health and disease.

Given the existence of AHL-mediated QS networks in the oral microbial environment, studies have focused on AHL-related interactions in oral commensal microorganisms (16). Researchers have previously assumed that Gram-negative periodontal organisms lack N-acyl HSL-dependent signalling systems (22). To determine whether AHL-related systems exist, one study investigated representative Gram-negative bacteria in the oral environment for a well-known putative third family of homoserine lactone synthetase S (HdtS) by introducing the genomic libraries of P. gingivalis W50 DNA into Escherichia coli strains containing either the lux-based AHLs reporter pSB401 or pSB1075 (23). These synthases tested negative for each of the transformants required for bioluminescence and the characteristics accompanying satellitism (15). Bioluminescence and satellitism are indicative tests commonly used to identify QS activity, specifically the presence and functionality of AHL synthases. The negative test result suggested that the transformants did not produce or respond to AHLs, implying that P. gingivalis likely lacked an AHL-based QS system. Despite the lack of evidence of an AHL-related system in P. gingivalis, researchers have investigated the influence of synthetic N-acyl HSLs on protein growth and production in P. gingivalis. They demonstrated that synthetic AHLs (N-tetradecanoyl HSL and acyl-CoA) completely and dose-dependently inhibit the growth of P. gingivalis strains and alter their SDS-PAGE expression profiles (24). Thus, P. gingivalis possesses a specific receptor that operates as an N-acyl HSL transcriptional activator and receives AHLs, complying with a mechanism similar to that of other Gram-negative bacteria (25). P. gingivalis W8326 was later found to contain an open reading frame with a 25% identity and 48% amino acid similarity to HdtS, indicating the presence of at least one putative AHL synthase in P. gingivalis W8326 (24).

Two other critical proteins were observed: LuxI, which catalyses the acylation and lactonisation reactions between the substrates S-adenosylmethionine and hexanoyl-ACP and then synthesises AHLs, and LuxR, which is the cytoplasmic receptor for AHLs and a transcriptional activator of the virulence-related operon (14). P. gingivalis was found to contain a LuxR homologue, Community Development and Hemin Regulator, which controls the transcription of the hmu operon responsible for iron/hemin uptake (26, 27). Whole-genome sequencing analysis showed that Citrobacter amalonaticus (C. amalonaticus) possesses the QS signalling synthase gene of a LuxI/LuxR functional pair clustered on the same chromosome.

Major Gram-negative periodontal pathogens, such as P. gingivalis and A. actinomycetemcomitans, utilise AHLs to regulate dental biofilm development. Gram-positive oral bacteria cannot produce AHLs but possess LuxR homologues, known as LuxR orphans, which can interact with QS molecules produced by other microorganisms in the environment (28). For example, a gene that belongs to the LuxR family of regulatory proteins has been predicted in the genome of Streptococcus mutans (29). Staphylococcus aureus can respond to OC₁₂-HSL produced by Pseudomonas aeruginosa (P. aeruginosa) in a saturable and specific manner, thus inhibiting the production of exotoxins and enhancing the expression of protein A, an important surface protein involved in several virulence mechanisms (30).

Even though earlier assumptions suggested a lack of AHL-dependent signalling in certain oral bacteria like P. gingivalis, recent studies have demonstrated the presence of AHL receptors and potential synthase genes. AHLs differentially regulate subsequent bacterial communication to express specific gene sets, ultimately triggering behavioural changes (15, 24). Evidently, the AHL-regulated QS system plays a crucial role in the coordination of behaviours among oral bacteria, influencing biofilm formation and pathogenicity. This process can aggravate oral biofilm-related diseases.

In addition to being integral components of bacterial QS systems, AHLs exert biological effects on human cells and influence oral diseases (Table 2). N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL, OC12-HSL) is secreted by P. aeruginosa, a Gram-negative opportunistic pathogen detected in periapical disease and periodontitis. OdDHL helps construct a niche suitable for P. aeruginosa invasion and reproduction, further aggravating bone destruction. OdDHL can regulate osteoblast metabolism (apoptosis and differentiation) by mediating intracellular calcium [(Ca2⁺)_i] fluctuations and spatial correlation in preosteoblastic MC3T3-E1 cells. Different concentrations of OdDHL trigger opposing osteoblast fates. At 50 μM, OdDHL inhibits osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein, and osteocalcin. Additionally, it elevated Ca2⁺_i in spatially autocorrelated osteoblasts. At 30 μm, OdDHL promoted osteoblast differentiation and apoptosis. Thus, AHLs affect osteoblast metabolism, the most important biological process for maintaining bone mass (35). Moreover, OdDHL plays a positive role in antitumor activity (36). Structure-activity relationship studies have found that C₁₂-HSL exhibits antiproliferative effects against human oral squamous carcinoma cells derived from gingival carcinoma (Ca9-22 cells) and tongue cancer (human tongue squamous carcinoma cells) as well as radiation-sensitising effects against Ca9-22 cells (34). AHLs not only modulate bacterial QS but also exert profound effects on human host cells, influencing processes critical to oral health such as osteoblast metabolism and bone homeostasis. This regulation of osteoblast activity highlights its potential impact on bone integrity in oral diseases like periodontitis. Additionally, the antiproliferative and radiation-sensitizing effects on oral squamous carcinoma cells suggest therapeutic avenues for cancer treatment. Generally, these findings underscore the multifaceted role of AHLs in oral health and disease.

AHL synthesis inhibitors disrupt the production of signalling molecules. Some natural products inhibit AHLs' biosynthesis, such as halogenated furanones from the marine alga Delisea pulchra (37). New bacterial species related to marine organisms were isolated and characterised as potential candidates for antimicrobial and antibiofilm treatments. Through AHL-based QS, bromoageliferin and oroidin, which are produced by marine organisms, trigger biofilm detachment in Gram-negative bacteria (38, 39).

A variety of chemical compounds have been investigated for their quorum quenching functions such as 4-nitro-pyridine-N-oxide from garlic cloves (40). However, these compounds have toxic and carcinogenic effects and offer poor stability in aqueous solutions. These factors restrict their use as antimicrobials (41). Macrolide antibiotics have been found to modulate AHLs' biosynthesis at non-lethal concentrations, though their precise mechanism of inhibition remains unclear (42). Additionally, pyrimidinone compounds have been explored as potential antagonists of AHLs, with a patent applied for their use in treating periodontal disease in the oral cavity (43). Pyrimidinones effectively inhibit C₆-HSL and C₈-HSL synthesis, toxin production by Burkholderia glumae, and biofilm formation by P. aeruginosa and P. gingivalis.

Analogues of AHLs mimic the structure of natural AHLs and competitively bind to the QS receptors, preventing the actual AHLs from activating the signalling pathway. Treatment with synthetic N-acyl HSL analogues inhibits biofilm formation by P. gingivalis, a primary etiological agent of periodontal disease. Treatment with 100 µM C₆-HSL and C₁₂-HSL analogues remarkably decreased P. gingivalis biofilm formation in a quantitative and structural (three-dimensional) manner, without inducing quantitative or qualitative differences in primary cell attachment. This suggests that the analogues affect the developmental stages of microcolonies and biofilm formation of P. gingivalis (31). However, this type of AHL inhibitors does not affect initial attachment or mature biofilms. The molecule 3-oxo-N-(2-oxocyclohexyl)-dodecanamide (3-oxo-N), a structural homologue of C₁₂-HSL, altered the ecological homeostasis of in vitro dental plaques by reducing the cariogenic potential through minimisation of lactic acid accumulation. However, it did not notably inhibit biofilm formation. Thus, 3-oxo-N is a potential compound for maintaining a healthy, non-cariogenic ecology in in vivo dental plaque. In addition to preventing caries, 3-oxo-N significantly alters species composition and ameliorates an unhealthy ecology by suppressing the maturation of Megasphaera micronuciformis and Solobacterium moorei, which were previously found in periodontal sites (32). The combined application of N-acyl HSL analogues and typical antibiotics reduces the viability of P. gingivalis cells in biofilms. A C₆-HSL analogue at a final concentration of 100 µmol L⁻¹ combined with 1 µg ml⁻¹ minocycline or cefuroxime exerted strong inhibitory effects on biofilm formation. This analogue reduced the thickness, volume, and matrix production of the biofilm, allowing easy antibiotic penetration into P. gingivalis colonies and demonstrating higher efficacy than a single application of antibiotics or analogues (25). Additionally, S-allyl-cysteine from garlic extracts, with a structure similar to that of AHLs, yielded excellent results in the treatment of a multi-species bacterial infection when combined with an antibiotic and/or antiseptic. This is especially effective for dental infections, including periodontal diseases such as gingivitis, periodontitis, pericoronitis, peri-implantitis, and related inflammation (44). AHL analogues of reduced the required antibiotic concentrations and treatment durations for controlling periodontal biofilm growth. This finding suggests a novel therapy for the treatment of periodontal diseases without the adverse effects of antibiotics or bacterial tolerance to antibiotics.

A previous study evaluated and improved the cytotoxicity of acridine-based AHL analogues in radiosensitizing human oral squamous carcinoma cells and human tongue squamous carcinoma cells. AHL analogues induce G2/M phase arrest and polyploidy. Synergised with X-irradiation, AHL analogues also inhibit the clonogenic survival of human tongue squamous carcinoma cells, which is related to mitotic failure following enhanced expression of Aurora A and B. Active AHL analogues can suppress the growth of human tongue squamous carcinoma cells and promote radiosensitisation (33). Thus, AHL analogues are considered new druggable targets and effective chemotherapeutic medicines. They can be used in treating human oral squamous carcinoma cells and in conjunction with radiotherapy as a postoperative treatment for long-term control.

AHL analogues show significant potential as innovative therapeutic agents for controlling bacterial biofilm formation, enhancing antibiotic efficacy, and serving as chemotherapeutic agents, particularly in the treatment of periodontal diseases and oral squamous carcinoma. Further research is necessary to fully understand their benefits and optimize their application in clinical settings.

AHL enzymatic degraders break down AHLs. They consist of three main types: lactonases, which target the lactone bond; acylases, which target the amide linkage; and oxidoreductases, which target the acyl chain. Gram-positive bacterial and eukaryotic (e.g., plant) cells produce enzymes such as lactonases and acylases that break down AHLs (45). Among these enzymes, lactonases are the most widely characterised and have been the focus of recent research. AHL lactonases hydrolytically cleave the ester bond of the HSL ring to form a homologous AHL derivative. The AHL lactonase Aii20j was effective against multi-species biofilms formed by several oral pathogens. It also significantly inhibited biofilm formation in in vitro oral biofilm models inoculated with saliva samples from healthy individuals and patients with oral diseases (21). Another AHL lactonase, est816, was shown to inhibit A. actinomycetemcomitans biofilm formation and virulence release, resulting in anti-inflammatory effects and smoothing periodontitis in rats (46). These findings demonstrate the potential of AHL enzymatic degraders as a promising biocontrol strategy to mitigate antimicrobial resistance. These enzymes—particularly lactonases—are capable of impairing the signaling pathways essential for biofilm formation and virulence expression. The demonstrated effectiveness of lactonases, such as Aii20j and est816, in inhibiting biofilm formation and reducing pathogenicity in both single-species and multi-species settings highlights their relevance in treating oral diseases.

Overall, by targeting bacterial communication specifically, these AHL inhibitors offer a precise approach to controlling bacterial infections without fostering resistant phenotypes, positioning them as innovative tools in combating microbial resistance and enhancing dental health.

Although existing analytical methods are most commonly used for detecting AHLs in biological and clinical samples, quantifying a small number of AHLs in culture media remains challenging due to several limitations. Hence, it is difficult to verify and quantify AHLs in oral samples and monospecies culture supernatants. A study in 2001 indicated that no A. actinomycetemcomitans strain could produce the tested AHLs (22). Previous reporters were primarily based on P. aeruginosa and Vibrio spp., which are structurally distinct from those found in oral bacteria. Moreover, only a few oral species have been tested, and they are not wholly representative of the complex multi-species biofilms in the oral cavity. Thus, identifying specific species in the AHL-mediated QS system is both challenging and important (47).

A thorough schematic representation of the AHL-related QS pathway in common oral bacteria remains unknown. Previous studies have mainly focused on the association between AHLs and P. gingivalis. Evidence has shown that the HdtS-CdhR system in P. gingivalis is similar to the LuxI-LuxR system. Also, C₈-HSL was detected in P. gingivalis laboratory samples. Thus, researchers proposed the following hypothesis: AHL-regulated QS system inherently controls the expression of specific virulence factors in P. gingivalis. These factors are implicated in the aetiology of human periodontal disease due to the virulence associated with the elaboration of cysteine proteases, Arg-gingipain (Rgp), and Lys-gingipain (Kgp) (2, 3). Whether AHLs affect the biomechanism of the expression of these main virulence genes through the QS system should be explored.

In addition to P. gingivalis, specific AHLs produced by other major Gram-negative oral bacteria in the biofilm matrix were not detected. These AHLs are also critical, as cognate AHLs can activate QS circuits, and non-cognate AHLs can inhibit the same receptor with similar structures (16). Therefore, future studies should conduct separate qualitative detections of various oral pathogens and consider the complexity of oral microorganisms when building oral biofilm models.

Research has demonstrated that AHLs indirectly inhibit immunosuppressive cytokines and chemokines and trigger the immunostimulating ones (48), by regulating the biological behaviours of oral pathogens. Thses pathogens use cell envelopes and exoproduct virulence to attach to the surface, continuously degrading host cell tissues and immune effector molecules and ultimately invading epithelial cells (49–51). No evidence has been found regarding the relationship between AHLs and immune-related inflammatory diseases extending into the oral environment. Monitoring the dynamic process of AHLs within cells is challenging. Varying concentrations of AHLs in immunocytes results in contrasting effects. Therefore, investigating the role of AHLs in either suppressing or stimulating cytokines and chemokines in oral inflammatory diseases is necessary. Understanding the correlation between AHLs and inflammatory mediators is crucial for further developing strategies involving AHL analogues or antagonists.